Field of the Invention
This invention relates to DNA amplification methods including improved real-time PCR detection methods, for the detection of Candidatus Liberibacter species from citrus and psyllid hosts. It also relates to novel DNA sequences, novel primers and probes made from the novel DNA sequences, and to kits containing said primers and reagents for the DNA amplification methods for the detection of Candidatus Liberibacter species.
Description of the Related Art
Citrus huanglongbing (HLB), also known as citrus greening, is a destructive disease that was first noted in the early 20th century in China (Zhao, Proc. Intl. Soc. Citriculture I, 466-469, 1981). This disease has spread throughout the global citrus producing regions, and has recently invaded North America, with first detection in Florida in 2005 (Knighten et al., USDA Departmental Release, Sep. 2, 2005). Three fastidious α-Proteobacteria species of Candidatus Liberibacter, Ca. L. asiaticus, Ca. L. americanus, and Ca. L. africanus (Bove, J. Plant Pathology, Volume 88, 7-37, 2006; Gottwald et al., Plant Health Program. Published online 2007) are associated with HLB. These bacteria have been shown to reside within sieve tube cells of infected plants (Tatineni et al., Phytopathology, Volume 98, 592-599, 2008) and to be vectored by psyllids, Diaphorina citri (Halbert and Manjunath, Florida Entomologist, Volume 87, 330-353, 2004) and Trioza erytreae (Bove et al., 2006 supra; McClean and Oberholzer, S. Afr. J. Agri. Sci, Volume 8, 297-298, 1965; McClean, Phytophylactica, Volume 6, 45-54, 1974).
Although HLB presents systemically, low titer and uneven distribution of the HLB bacteria within infected plants (Tatineni et al, 2008, supra; Teixeira et al., Mol. Cell. Probes, Volume 22, 139-150, 2008; Li et al., Phytopathology, Volume 99, 139-144, 2009) can make reliable detection difficult. As such, many methods have been developed including biological indexing using graft and dodder transmission (Gottwald et al., 2007, supra), light or electronmicroscopy (Bove, 2006, supra), loop-mediated isothermal amplification (Okuda et al., Plant Disease, Volume 89, 705-711, 2005), polymerase chain reaction (PCR) (Jagoueix et al., Mol. Cell. Probes, Volume 10, 43-50, 1996; Hung et al., J. Phytopathology, Volume 147, 599-604, 1999; Tian et al., Proc. Conf. Int. Org. Cirus Virol., Volume 13, 252-257, 1996), and real-time PCR (Teixeria et al., Mol. Cell. Probes, Volume 22, 139-150, 2008; Li et al., Phytopathology, Volume 99, 139-144, 2009; Li et al., Plant Disease, Volume 92, 854-861, 2008; Li et al., Plant Disease, Volume 91, 51-58, 2007; Li et al., J. Microbiol. Methods, Volume 66, 104-115, 2006; Wang et al., Plant Pathology, Volume 55, 630-638, 2006) to detect these Ca. Liberibacter bacteria. However, these detection methods are typically diagnostic only after HLB associated phenotypic symptoms are observable. Furthermore, the etiology of HLB remains, to a large extent, undefined.
Currently real-time PCR has become the preferred detection method of Liberibacter species (Teixeira et al., 2008, supra; Li et al., 2009, supra; Li et al., 2008, supra; Li et al., 2007, supra; Li et al., 2006, supra; Wang et al., 2006, supra). Relative to conventional PCR, real-time PCR offers both sensitive and rapid detection of these bacteria. Real-time PCR is reported to increase the sensitivity for Liberibacter detection by 10 times relative to nested PCR (Teixeira et al., 2008, supra) and 100 to 1,000 times relative to conventional PCR (Teixeira et al., 2008, supra; Wang et al., 2006, supra) for these bacteria. These real-time PCR methods target genes with low copy number; three copy 16S rDNA (Li et al., 2006 supra), single copy β-operon (Teixeira et al, 2008, supra) or single copy elongation factor Ts (EF-Ts) (Lin et al., J. Microbiol. Methods, Volume 81, 17-25, 2010). The reported real-time PCR low threshold limits are approximately ten gene copies for 16S rDNA and β-operon methods (Teixeira et al., 2008, supra; Li et al., 2008, supra), with elongation factor Ts (single closed tube dual primer) reporting single gene copy detectability (Lin et al., 2010, supra). However, current PCR detection methods can miss the targeted DNA for amplification because the Ca. Liberibacter bacteria can exist at extremely low titer in their host plant and insect.
While various methods for detecting Ca. Liberibacter species have been developed, there remains a need in the art for a method for detecting extremely low titer levels of Ca. Liberibacter species. The present invention described below includes a sensitive and rapid new method for detecting Ca. Liberibacter species as well novel DNA sequences; and primers and probes made from these novel sequences which are different from related art methods and primers.